Treating disorders characterized by excessive cell proliferation with sclareolide

ABSTRACT

A method for treating a disorder characterized by excessive cell proliferation in a patient by administering to the patient a therapeutically effective amount of sclareolide.

(+) Sclareolide 3aR-(3aα, 5aβ, 9aα,9bα)!-decahydro-3a,6.6.9a-tetramethylnaphtho 2,1-b! furane-2(1H)-one isa natural bicyclic terpenoid which is found for example in tobacco. II.Kaneko, Agr. Biol. Chem. 35(9): 1461 (1971). (+) Sclareolide has thefollowing structure: ##STR1##

(+) Sclareolide is known for increasing or developing the organolepticproperties of food products. See for example U.S. Pat. Nos. 4,917,913;4,960,603; 4,966,783; 4,988,527; and 4,999,207. This compound was usedas a perfume for cigarettes (Japanese Patent No. 60,123,483) and as anadditive to eliminate the bitter taste of coffee (U.S. Pat. No.4,988,532).

However, to the filer's knowledge, (+) sclareolide has never been usedor presented as a pharmacologically active compound.

The present invention relates to a method for inhibiting a diseasecharacterized by excessive proliferation of cells in a patient (forexample, a mammal such as man) including the administration to thepatient of a therapeutically effective quantity of (+) sclareolide.

In one configuration, the patient is suffering from a disease associatedwith excessive proliferation of benign cells (i.e. non-malignant).Examples of such diseases are fibrosis, benign prostate hyperplasia,atherosclerosis, restenosis, glomulerosclerosis, cheloid, psoriasis, andother diseases of the skin and non-malignant neoplastic diseases.

In another configuration, the patient is suffering from a diseaseassociated with an excessive proliferation of malignant cells (forexample, cancer). Examples of such diseases are adenomas, carcinomas,cancers found in the prostate, the lungs, the liver, the pancreas, thebrain, the breast, and the skin, as well as leukemia.

The therapeutically effective quantity depends upon the conditiontreated and the route of administration chosen, as well as the specificactivity of the compound used and will be decided finally by theattending physician or veterinarian. (|) Sclareolide is administered inquantities of 0.1 to 500 mg/kg of body weight (for example, 1 to 100mg/kg of body weight).

While it is possible to administer (+) sclareolide in the form of a pureor substantially pure compound, this product may also be presented inthe form of a formula, a preparation, or a pharmaceutical composition.The formulas to be used in the present invention, both for human beingsand animals, include the (+) sclareolide associated with one or moreacceptable pharmaceutical vehicles of the latter and optionally othertherapeutic agents. The vehicle must be "acceptable," i.e. compatiblewith the active ingredient(s) of the formula and not noxious to thesubject to be treated.

Formulas may be conveniently presented in the form of a single dosageand may be prepared by any of the well known methods in the art ofpharmacy. All methods include the phase comprising of bringing (+)sclareolide into an association with a vehicle which may contain one ormore auxiliary agents. In general, compositions intended for themanufacture of tablets (for example for oral administration) or ofpowders are prepared by thorough and uniform mixing of (+) sclareolidewith the finely divided solid vehicles, followed if necessary, as in thecase of tablets, by placing the product into a mold to give it thedesired size and shape.

Compositions suitable for parenteral administration (for example,subcutaneous, intravenous, or intramuscular) moreover includeconveniently sterile aqueous solutions in which the (+) sclareolide issoluble. Preferably the solutions are isotonic with the blood of thesubject to be treated. These compositions may be conveniently preparedby dissolving the (+) sclareolide in an aqueous solution of this type,said solution subsequently being rendered sterile. The composition maybe presented in single or multiple dose containers, for example sealedvials.

Consequently the invention likewise relates to pharmaceuticalcompositions including as an active substance (+) sclareolide inassociation with one or more acceptable pharmaceutical vehicles.

Another subject matter of the invention comprises claiming the use of(+) sclareolide for the preparation of a medication intended for thetreatment of diseases characterized by the excessive proliferation ofcells. Examples of such diseases are fibrosis, benign prostatehyperplasia, atherosclerosis, restenosis, glomerulosclerosis, cheloid,psoriasis, and other diseases of the skin and non-malignant neoplasticdiseases, adenomas, carcinomas, cancers found in the prostate, thelungs, the liver, the pancreas, the brain, the breast, and the skin, aswell as leukemia.

More particularly, the invention relates to (+) sclareolide as amedication in treatment methods.

Other characteristics and advantages of the present invention willappear in the course of the detailed description of the invention and inthe claims.

It is considered that a specialist may use the present invention in itsentirety based upon the present description. The following specificconfigurations should as a result be interpreted as purely illustrativeand not limiting the rest of the disclosure, regardless of its nature.

Unless they are otherwise defined, all of the technical and scientificterms used here have the same meaning as that currently understood by aperson of ordinary competence in that field of technology of which thisinvention is a part. Likewise, all publications, patent applications,and other references cited herein are incorporated by reference.

(+) Sclareolide

(+) Sclareolide is available from a specific number of commercialsources, for example Aldrich Chemical Co., St. Louis, Mo. (+)Sclareolide may likewise be prepared by synthesis, for example from (-)sclareol (Aldrich Chemical Co.) or homophamesylic acid. See for exampleCoste Maniere et al., Tetrahedron Letters, 29(9):1017 (1988), Mantres etal., Tetrahedron Letters 34(4):629 (1993); German Patents No. DE4,301,555 and DE 3,942,358 and PCT Application No. WO 93/21,174.

Inhibition of the proliferation of benign prostate hyperplasia (HPB)

The solutions of (+) sclareolide are prepared by dilution withdimethylsulfoxide (DMSO) (0.5%). The culture medium used was a minimumessential medium (MEM, Gibco, Taisley, United Kingdom) without any serumbut to which L-glutamine (0.6 mg/ml, Gibco), gentamycin (40 μg/ml,Gibco), penicillin (100 IV/ml, Gibco), and streptomycin (100 μg/ml,Gibco), has been added. Tritiated thymidine (dThd: spec. act. 48Ci/mmole) was obtained from Amersham (Little Chalfont, United Kingdom).The solutions to be added to the incubation medium were prepared at thetime of use by appropriate dilution with the MEM. The HPB tissue wasobtained from 10 patients (age range 56-80 years; average age+/-standarddeviation: 68+/-12) having prostates not previously treated, which hadundergone an open tetropubic prostatectomy. The specimens were receivedfresh from the operating room in the MFM kept at 4° C. for 1-3 hoursbefore being treated for organ culture as presented in detail in Y.Launoit et al., The Prostate, 13:143 (1988). For each HPB specimen, eachexperimental condition was determined by means of a series of 10 samplesof tissue (0.5-1 mm³) placed in a petri dish (3 cm in diameter, Gibco)containing 2.5 ml of MEM medium. These cultures were incubated in acontrol medium or in a medium to which had been added either 10 nM ofEpithelial Growth Factor (FCE) or 10 nM of basic Fibroblast GrowthFactor (FCFb) with or without 10⁴ M (final concentration) of (+)sclareolide. FCE or FCFb were added at the beginning of the culture. The(+) sclareolide was added 24 hours after the FCE or the FCFb. Thecultures were immersed in the fixer EFA (96° ethanol, 70% by volume,neutral lormol, 25% by volume, acetic acid, 5% by volume) at the 72ndhour after the beginning of the culture, i.e. at the 48th hour after theaddition of the (|) sclareolide Tritiated thymidine (2 μCi/ml MEM) wasadded to the culture medium four times, namely at 36 hours, 24 hours, 12hours and 1 hour before fixation.

The method of tagging with tritiated thymidine made it possible toestimate the tagging indexes with thymidine (IMT). The IMT representsthe percentage of cells involved in the S phase of the cell cycle. Thisindex therefore represents an indirect estimate of the rate ofproliferation in a given tissue. The methodology used here is identicalto that presented in detail in Y. Launoit et al., The Prostate, 13: 143(1988). The IMT was determined as follows. Two sections of each specimenwere cut of the HPB. The IMT was determined separately in the stromaland epithelial compartments of each HPB. For this purpose, the IMTcounts were carried out on 300-800 stromal cells and 300-800 epithelialcells per section of HPB. Thus, in sum, 600-1,600 stromal cells and600-1,600 epithelial cells were counted per specimen of HPB and in sum,6,000-16,000 stromal cells and 6,000-16,000 epithelial cells werecounted per experimental condition. The nuclear tagging was consideredpositive when a node was covered by a number of grains of silver whoseaverage value was 10 times higher than that of the background.

It was noted that the (+) sclareolide inhibited the proliferation oforganotypical cells of HPB simulated by the FCFb at 70% and inhibitedthe proliferation of cells of HPB stimulated by the Epithelial GrowthFactor at 60% for a concentration of 10⁴ M.

Inhibition of the proliferation of fibroblasts

A confluent culture of mouse embryo cells (CFS) was used to determinethe proliferation of fibroblasts by measuring the incorporation of ³ H!methyl thymidine into the DNA of these cells. The CES were brought intosuspension in a Dulbecco modified essential medium (DMEM: Gibco). TheDMEM in this test comprised 1 g/l of glucose, 100 μg/ml of penicillin,100 μg/ml of streptomycin, and 10% of fetal calf serum. 24 cavityculture plates were filled with 500 μl of the suspension (70,000cells/cavity) and maintained for 24 hours in an incubator at 37° C. at5% CO₂.

The next day, the culture medium in each cavity was replaced by 500 μlof DMEM containing 0.5% fetal calf serum for the purpose of reducing therate of reproduction and keeping the cells in a stationary phase. Thenext day the culture medium was replaced (1 μl for each cavity) and thecells were stimulated with FCFb with or without a solution of varyingconcentrations of (+) sclareolide. The solutions of (+) sclareolide wereprepared by aqueous dilution with DMSO. The next day ³ H! methylthymidine (1 μCi/ml) was added and the culture plates were kept for fourhours at 37° C. The incubation medium was subsequently removed and 1 mlof cooled 10% trichloracetic acid was added (ATC, Sigma Chemical). 30minutes later at 4° C. the ATC was removed, the culture plates wererinsed with water and 500 μl of 0.3 m NaOH were added to each cavity.The culture plates were kept at 4° C. for one night. The next day thecultures of each cavity were transferred to scintillation vials. TheNaOH was neutralized with 500 μl of HCl (0.3 N) and the radioactivity ofthe scintillation vials was measured by means of a Beckmannscintillation counter (Model No. L560000SG).

It was found that the (+) sclareolide inhibited the proliferation offibroblasts stimulated by the FCFb 50% for a concentration of 10⁻⁷ M and70% for a concentration of 10⁻⁵ M.

It should be understood that although the invention was presented with adetailed description, the purpose of the preceding description was toillustrate and not to limit the field of application of the invention,which is defined by the field of application of the enclosed claims.Other aspects, advantages, and modifications are given in the claims.

We claim:
 1. A method of treating excessive proliferation of benign andmalignant cells sensitive to the compound below in mammals comprisingadministering to mammals in need thereof of an amount of (+) sclareolidesufficient to reduce proliferation of benign and malignant cells.
 2. Amethod according to claim 1, characterized in that said proliferation ofcells is benign.
 3. A method according to claim 2, characterized in thatsaid (+) sclareolide is administered by parenteral route.
 4. A methodaccording to claim 2, characterized in that said (+) sclareolide isadministered by subcutaneous route.
 5. A method according to claim 2,characterized in that said (+) sclareolide is administered orally.
 6. Amethod according to claim 1, characterized in that said disease isfibrosis.
 7. A method according to claim 6, characterized in that said(+) sclareolide is administered by parenteral route.
 8. A methodaccording to claim 6, characterized in that said (+) sclareolide isadministered by subcutaneous route.
 9. A method according to claim 6,characterized in that said (|) sclareolide is administered orally.
 10. Amethod according to claim 1, characterized in that said disease is abenign prostate hyperplasia.
 11. A method according to claim 10,characterized in that said (+) sclareolide is administered by parenteralroute.
 12. A method according to claim 10, characterized in that said(+) sclareolide is administered by subcutaneous route.
 13. A methodaccording to claim 10, characterized in that said (+) sclareolide isadministered orally.
 14. A method according to claim 1, characterized inthat said (+) sclareolide is administered by parenteral route.
 15. Amethod according to claim 1, characterized in that said (+) sclareolideis administered by subcutaneous route.
 16. A method according to claim1, characterized in that said (+) sclareolide is administered orally.